Figure 5.

sLZIP binds to the enhancer region of FABP4. (a) Analysis of the FABP4 enhancer sequence by Transcription Element Search System and AliBaba2. (b) Recombinant His-sLZIP fusion protein (3 μg) was incubated with γ-³²P-labeled oligonucleotide probes containing the putative CREB-binding element. (c) γ-³²P-labeled oligonucleotide probes were incubated with increasing amounts of purified His-sLZIP. A competition assay was performed using a 100-fold molar excess of unlabeled CRE oligonucleotide probes. His antibody was added to the reaction mixture for a supershift assay. (d) His-sLZIP protein was subjected to an EMSA using WT and CRE mutants (TGATGTCA→TGGGGTCA). (e) C3H10T1/2 cells were infected with adenoviral HA-sLZIP and differentiated into adipocytes. After 8 days from post-induction, ChIP assays were performed using an antibody specific for HA-sLZIP. Input or immunoprecipitated chromatin was subjected to a PCR as a control for variations. (f) 293T cells were transfected with FABP4 enhancer-Luc, β-galactosidase, PPARγ₂, and various amounts of sLZIP (0.1, 0.25, and 0.5 μg). Following transfection, cells were stimulated with 10 μM Rosi for 24 h. Luciferase activities were normalized to the β-galactosidase activity and presented as the relative activity. All experiments were performed in triplicate and the bar represents the mean±S.D.